Curr. Issues Mol. Biol. 14: 9-26. Ribonucleotide Reductase as a Target to ControlOnline Apicomplexan journal at http://www.cimb.org Diseases 9 Ribonucleotide Reductase as a Target to Control Apicomplexan Diseases

James B. Munro and Joana C. Silva* hosts/vectors and transition between life cycle stages is dependent upon a diverse array of environmental cues. Department of Microbiology and Immunology and Institute The biological characteristics that differentiate for Genome Sciences, University of Maryland School of apicomplexans from their vertebrate hosts have often been Medicine, Baltimore, MD 21201, USA considered optimal targets of new therapeutics to control these eukaryotic pathogens. Alternatively, essential and strongly conserved proteins can be targeted, provided that Abstract they differ from their vertebrate homologs in such a way that Malaria is caused by species in the apicomplexan genus minimizes potential cross-reaction and toxicity. Plasmodium, which infect hundreds of millions of people The enzyme ribonucleotide reductase (RNR) is one each year and kill close to one million. While malaria is such example. RNR utilizes free radical chemistry to catalyze the most notorious of the apicomplexan-caused diseases, the reduction of ribonucleotides to deoxyribonucleotides other members of the eukaryotic phylum Apicomplexa (Thelander and Reichard, 1979; Reichard, 1988). It provides are responsible for additional, albeit less well-known, the only de novo means of generating the essential building diseases in humans, economically important livestock, and blocks for DNA replication and repair across all domains a variety of other vertebrates. Diseases such as babesiosis of life and, as such, it is the rate-limiting step in DNA (hemolytic anemia), theileriosis and East Coast Fever, synthesis (Jordan and Reichard, 1998; Lundin et al., 2009). cryptosporidiosis, and toxoplasmosis are caused by the Additionally, RNR is critical for maintaining a balanced pool apicomplexans Babesia, Theileria, Cryptosporidium and of DNA precursors during chromosome replication (Herrick Toxoplasma, respectively. In addition to the loss of human and Sclavi, 2007). An unbalanced deoxyribonucleotide life, these diseases are responsible for losses of billions triphosphate pool may lead to an increase in mutation and of dollars annually. Hence, the research into new drug disease (Lin and Elford, 1980; Reichard, 1988; Chabes et targets remains a high priority. Ribonucleotide reductase al., 2003; Wheeler et al., 2005; Gon et al., 2006; Mathews, (RNR) is an essential enzyme found in all domains of 2006; Kumar et al., 2010). life. It is the only means by which de novo synthesis of Here we review the chemotherapeutic methods used to deoxyribonucleotides occurs, without which DNA replication inhibit the essential enzyme RNR, with particular emphasis and repair cannot proceed. RNR has long been the target of on the novel RNR R2_e2 subunit recently identifed in antiviral, antibacterial and anti-cancer therapeutics. Herein, apicomplexan parasites (Munro et al., submitted) and on the we review the chemotherapeutic methods used to inhibit malaria-causing genus Plasmodium. The R2_e2 subunit is RNR, with particular emphasis on the role of RNR inhibition unique to the Apicomplexa and as such, it can potentially be in Apicomplexa, and in light of the novel RNR R2_e2 subunit used to specifcally target apicomplexan pathogens. recently identifed in apicomplexan parasites. Apicomplexan parasites are responsible for devastating Introduction infectious diseases The Apicomplexa are a group of single-celled, eukaryotic The phylum Apicomplexa consists of more than 4,000 organisms that, together with the ciliates and dinofagelates, described species (Levine, 1988), many of which are of form the major lineages in the Alveolates. All Apicomplexa, medical, agricultural, and economic importance and whose save the predatory fagellates Colpodellida, are pathogenetic, adverse impact on human society cannot be overstated. obligate, intracellular parasites (Adl et al., 2005; Morrison, Among the most notorious are Plasmodium, Babesia, 2009). They are characterized by the presence of an apical Theileria, Cryptosporidium, and Toxoplasma the causative complex, a structure involved in host-cell invasion, which is agents of malaria, babesiosis, theileriosis and East Coast located at the anterior end of the cell. Most apicomplexans fever, cryptosporidiosis, and toxoplasmosis, respectively. also possess a specialized organelle called the apicoplast, They are responsible for causing millions of human deaths a secondary endosymbiotic plastid believed to be of red and billions of dollars in productivity and material losses algal origin (Blanchard and Hicks, 1999; Fast et al., 2001; each year (Sachs and Malaney, 2002; Corso et al., 2003; Janouškovec et al., 2010). Within the apicoplast occur Rowe et al., 2006; Spielman, 2009). Currently, fve species processes essential for the parasite's survival, such as of Plasmodium are known to cause malaria in humans, P. heme and lipid biosynthesis. Another defning characteristic falciparum, P. knowlesi, P. malariae, P. ovale, and P. vivax of apicomplexans is their inability to synthesize purine rings (Rougemont et al., 2004; Singh et al., 2004; Cox-Singh et de novo and hence their need to salvage exogenous purines al., 2008), of which P. falciparum is the most deadly and via a variety of different pathways (Booden and Hull, 1973; P. vivax the most geographically widespread. The life cycle Chaudhary et al., 2004; de Koning et al., 2005; Cassera of Plasmodium alternates between a vertebrate host and et al., 2008; Madrid et al., 2008). These organisms have mosquito vector and involves four major developmental complex (indirect) life cycles, and they often exploit multiple stages in the vertebrate host: sporozoites, merozoites, trophozoites, and gametocytes (Bledsoe, 2005; Brown and Catteruccia, 2006). *Corresponding author: [email protected]

Horizon Scientifc Press. http://www.horizonpress.com 10 Munro and Silva

Prioritization of apicomplexan drug targets been proposed as a means to combat the emergence of There is currently no fully effcacious vaccine on the market drug resistance (Durand et al., 2008). Genes with high rates against any apicomplexan species and drug treatment is of nonsynonymous changes have been associated with the method of choice in the management of apicomplexan drug resistance in P. vivax (Dharia et al., 2010), and may be diseases. Modern approaches to drug design, made responsible for vaccine evasion in P. falciparum (Takala and possible with the advent of genome sequencing, emphasize Plowe, 2009). In contrast, evolutionary patterning focuses defning and targeting metabolic and molecular differences on fnding and targeting protein residues that are under between host and parasite to avoid host side effects (Croft, strong purifying selection, which will in principle reduce the 1997). This may be achieved with the selective targeting of instances of drug resistance mutations. parasite-specifc enzymes or by targeting those which are The tremendous health burden imposed by malaria highly divergent or have distinct binding sites and are thus has made Plasmodium the primary target for many of these suffciently different to be selectively targeted (Coombs, approaches. Despite a diversifed arsenal of potential tools 1999; Cerqueira et al., 2007). Also important is that the to combat malarial infection, multiple drug resistance to protein target be essential to the growth, reproduction, or existing anti-malarial compounds is becoming increasingly survival of the parasite. Finally, knowledge of the gene common in Plasmodium (Greenwood and Mutabingwa, expression pattern of potential target proteins is necessary to 2002; Anderson, 2009; Bustamante et al., 2009; Takala and link drug administration with the critical and relevant stages Plowe, 2009). A case in point is the antifolate drugs used to of the pathogen's life cycle. This is particularly pertinent to treat malaria. Antifolate drugs bind enzymes necessary for organisms characterized by differentially expressed, stage- folate biosynthesis, thus targeting essential precursors for specifc, and often stage-unique gene expression, such as purine and pyrimidine synthesis. Antifolates have been used apicomplexans (Coulson et al., 2004). It has been suggested against Plasmodium with success, but resistance to these that targeting the intraerythrocytic life stages of Plasmodium drugs has become widespread (Gregson and Plowe, 2005; (the ring forms, trophozoites, schizonts, and merozoites), Mkulama et al., 2008; Sridaran et al., 2010). Currently, the when clinical symptoms are manifested, is of particular primary treatment for malaria is based on artemisinin, which interest (Yeh and Altman, 2006). However, targeting multiple is administered in combination with other drugs in order to life stages, for example controlling both the liver and blood prevent, or delay, the onset of resistance. However, there stages of Plasmodium, may be more conducive to the are clear indications that artemisinin resistance is emerging ultimate goal of disease eradication (Alonso et al., 2011). (Chrubasik and Jacobson, 2010; Dondorp et al., 2010; Enserink, 2010; Fidock, 2010). Therefore, the development Current control strategies and challenges of new chemotherapeutic and prophylactic antimalarial Drug targets for control of apicomplexans have focused on drugs and vaccines remains a priority (Greenwood and parasite metabolic processes. These include processes Mutabingwa, 2002; Anderson, 2009; Bustamante et al., within the cytosol, mitochondrion, digestive vacuole, 2009; Takala and Plowe, 2009). synthesis of macromolecular and metabolic enzymes, and processes involved in membrane synthesis and signaling RNR classifcation and distribution (Olliaro and Yuthavong, 1999; Padmanaban, 2003; Fidock RNRs are classifed into one of three classes, I-III (Jordan et al., 2004; El Bissati et al., 2006). Enzymes in the purine et al., 1994; Reichard, 1997; Fontecave, 1998; Nordlund salvage pathways, essential to these pathogens, are also and Reichard, 2006). All three classes use a thiyl radical potential drug targets (Tracy and Sherman, 1972; Krug to remove the ribose OH-group. The distinction between et al., 1989; Parker et al., 2000; Gardner et al., 2002; these classes relies on differences in radical generation Raman and Balaram, 2004; Striepen et al., 2004; Ting chemistry and the cofactor needed to produce the organic et al., 2005; Downie et al., 2008), as are all proteins and radical. Class I RNRs are oxygen-dependent, typically processes related to the apicomplexan-specifc organelle, require a tyrosyl radical and a diiron center, and are the apicoplast (Foth et al., 2003; Sato and Wilson, 2005; characteristic of eukaryotes and common among bacteria. Wiesner et al., 2008; Lizundia et al., 2009). Antibiotics like Class II RNRs are indifferent to oxygen, form a thiyl radical doxycycline, which specifcally target plastid pathways and via adenosylcobalamin, and are characteristic of Archaea impair the expression of apicoplast genes, can be used and bacteria. Class III RNRs are anaerobic, form a glycyl to target apicomplexans (Ralph et al., 2001; Leander and radical using an iron-sulfur center in the presence of Keeling, 2003; Dahl et al., 2006). Apicoplast drug targets S-adenosylmethionine and reduced favodoxin, and are have included the apicoplast's metabolic pathways (e.g. also characteristic of Archaea and bacteria. DNA replication, transcription, protein translation, fatty acid Class I RNRs have been further categorized as class biosynthesis, and isoprenoid biosynthesis), or targeting Ia, Ib, or Ic. Class Ia is typical in eukaryotes and bacteria, proteins encoded by the host's nuclear genes that are while bacteria and Archaea primarily encode classes Ib and destined for the apicoplast (McFadden and Roos, 1999; Ic RNRs (Harder, 1993; Sjöberg, 2010). Class Ic RNRs Roos et al., 1999; Roos et al., 2002; Ralph et al., 2004; are further categorized as R2c proteins and while the White, 2004; Waller and McFadden, 2005; Dahl and R2-homolog R2lox proteins are described as “R2c-like”, Rosenthal, 2008; Prusty et al., 2010). they are not believed to form active RNR holoenzymes The availability of genome sequences from several (Högbom et al., 2004; Andersson and Högbom, 2009). species and isolates of Plasmodium and other Apicomplexa This classifcation, based on structural and chemical facilitates the identifcation of novel, potential drugs for the properties, has shortcomings since classes Ia and Ic are control of apicomplexan parasites (Doolan et al., 2003; Yeh not monophyletic clades, i.e., discrete, mutually exclusive et al., 2004; Carvalho and Ménard, 2005; Winzeler, 2008; groups, which contain a most recent common ancestor and Mu et al., 2010). For example, evolutionary patterning has all of its descendants. Instead, class Ia is polyphyletic and Ic Ribonucleotide Reductase as a Target to Control Apicomplexan Diseases 11 is paraphyletic. This problem was been addressed by Munro and two allosteric effector-binding sites. dATP and ATP bind et al. (submitted) (Figure 1) who recognized four distinct to the frst allosteric site, the A-site, and serve as inhibitor class I RNR clades, namely the eukaryotic-specifc clades and stimulator, respectively, while binding of dATP, ATP, R2_e1 and R2_e2, and the clades containing primarily dGTP and dTTP to the S-site determines substrate-binding archaeal and bacterial RNRs, namely R2_ab and R2c. The preference (Brown and Reichard, 1969; Reichard et al., study also revealed that the newly discovered clade R2_e2 2000). Allosteric regulation is accomplished by changes is unique to the Apicomplexa (Munro et al., submitted). In in the conformation of loop 2 which spans both the A- and fact, the most signifcant mammalian-infecting Apicomplexa S-sites, and which is determined by the molecule bound to genera, such as Plasmodium, Cryptosporidium, and the A-site (Reichard, 2010). Reviews and additional details Babesia, all encode one R2_e2 subunit. can be found in (Eriksson et al., 1979; Reichard et al., 2000; Reichard, 2002; Crona et al., 2010; Logan, 2011). Class Ia holoenzyme regulation and formation Subunit R2, the smaller subunit, contains an amino acid Apicomplexans encode Class Ia RNRs, which are thus the residue that harbors the organic radical (Nordlund et al., focus of this review. Class Ia RNR enzymes are composed 1990; Nordlund and Eklund, 1993). A long-range electron- of two distinct subunits, R1 and R2. Subunit R1, the larger coupled pathway connects the R2 radical to a cysteine of the subunits, contains a catalytic site (substrate binding) in R1 (the thiyl radical) via hydrogen-bonded amino acid residue side chains (Nordlund et al., 1990; Stubbe et al., 2003; Kolberg et al., 2004). Binding of the R2 subunit to R2lox the R1 subunit involves the C-terminus residues of the "
 

 R2 subunit interacting with a hydrophobic cleft in the R1 "
  
  subunit and it has been suggested that oligomerization of "

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   R1 is a prerequisite (Climent et al., 1992; Rova et al., 1999; "


! Uppsten et al., 2006). "
! 


 
 The most current model for RNR in eukaryotes suggests a holoenzyme with eight subunits and is of the form α6β2, R2c where alpha stands for the R1 subunit and beta for R2 "
 

 (Rofougaran et al., 2006). It has been proposed that in the "
  
   
 absence of the effectors dATP, ATP, dGTP and dTTP, the "

!!
   R1 subunit is an inactive monomer; however, once dTTP "

! or dGTP are bound to the S-site, an α2β2 heterodimer is formed (Ingemarson and Thelander, 1996). With increasing dATP concentration (which induces enzyme inhibition), R1 R2_ab monomers form dimmers and eventually inactive hexamers "
 

 (formation of intermediate tetramers remains in question), "

  while in the presence of the enzyme activator ATP, the "
!!
   holoenzyme adopts an α6β2 conformation (Fairman et al., "

! 2011).

Paralogous copies of the R2 subunit R2_e1 Many eukaryote genomes encode two or more distinct "
 !
   copies of the small R2 subunit (Lundin et al., 2009). For "

   
example, humans have the R2 and p53R2 paralogs "
!!
  
and S. cerevisiae the Y2 and Y4 paralogs. It is clear that "

! these copies have different functional roles, be it de novo creation of deoxyribonucleotides, maintenance of the R2_e2 deoxyribonucleotide pool, mitochondrial DNA replication, "
 !
  or DNA damage repair (Elledge and Davis, 1990; Huang "

  and Elledge, 1997; Tanaka et al., 2000; Lin et al., 2004; "
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   Bourdon et al., 2007). In such instances, a ββ' confguration "


! is believed to contribute to the active holoenzyme, although ββ and β'β' confgurations have been reported (Wang et al., 1997; Nguyen et al., 1999; Chabes et al., 2000; Ge et al., Figure 1. Unrooted phylogenetic relationships between the 2001; Guittet et al., 2001; Voegtli et al., 2001; Perlstein et RNR class Ia and Ic subunits and the R2lox R2 homolog al., 2005; Ortigosa et al., 2006). proteins (Munro et al., submitted). Class Ic includes the While most eukaryotes encode two R2 genes belonging R2c proteins; however, this classifcation proved to be to the typical eukaryotic clade R2_e1, apicomplexans encode paraphyletic as it failed to include the clade of proteins one R2_e1 subunit and one R2_e2 subunit. Toxoplasma now designated as R2_ab. Former class Ia proved to be appears to be an exception, as so far two R2_e1-encoding polyphyletic, including the novel R2_ab clade, which does genes have been identifed but no R2_e2 has been found in not share a recent common ancestor with the monophyletic its genome. R2_e1 and R2_e2 clade. Reference to the clades R2c, The conservation of functionally important R1 active R2_ab, R2_e1, and R2_e2 now allows for unambiguous site cysteines, and electron transfer cysteine and tyrosine reference to these RNR subunits. residues, as well as the conservation of R2 residues involved 12 Munro and Silva in iron binding, electron transport, free radical transfer, subunit had tumor-suppressing activity. Cao et al. (Cao et and the formation of the hydrophobic pocket around the al., 2003) utilized a recombinant adenovirus that encoded radical, implies that both eukaryotic pathogens and their and over-expressed the human R1 gene, which reduced hosts utilize the same free radical chemistry to synthesize proliferation of human colon adenocarcinoma cells, yet had deoxyribonucleotides (Hofer et al., 1997; Roshick et al., no effect on normal cells. On the other hand, inhibition of 2000; Akiyoshi et al., 2002; Shao et al., 2006). As such, the R2 subunit may have an antineoplastic effect, serving it would appear that targeting apicomplexan RNR by to inhibit and combat the development of cancer cells. chemotherapeutic means might have an adverse effect on Expression of R2 in conjunction with activated oncogenes the human host. This is not necessarily so. impacts a cell's malignant potential (Fan et al., 1998; Desai While prokaryotic and eukaryotic R1 and R2 subunits are et al., 2005). Overexpression of R2 is linked to increased highly conserved at, and around, the functionally important drug resistance and increased invasive potential in cancer residues (Chakrabarti et al., 1993; Sjöberg, 1997; Roshick et cells (Yen, 2003). al., 2000; Voegtli et al., 2001; Högbom et al., 2004; Högbom, RNR inhibition has been applied to the control of viruses 2010), there is considerable variation in the sequences at (Gaudreau et al., 1987; Moss et al., 1993; Bianchi et al., both the N- and C-termini (Ingram and Kinnaird, 1999). 1994; Szekeres et al., 1997; Robins, 1999), bacteria (Yang In particular, the eukaryotic orthodox R2 subunit, R2_e1, et al., 1997; Mdluli and Spigelman, 2006; Ericsson et al., presents distinct differences between apicomplexans and 2010; Lou and Zhang, 2010; Torrents and Sjöberg, 2010), mammals, including differences in key functional regions of and certain cancers (Cory, 1988; Nocentini, 1996; Gwilt the R2 protein; perhaps most notable are those differences and Tracewell, 1998). Because inhibition of RNR ceases, between C-terminal sequences (Bracchi-Ricard et al., or severely reduces, DNA replication, it has long been 2005). Furthermore, and more pertinent to this review, is considered an ideal target for the control of pathogens. As the fact that the apicomplexan-specifc R2 subunit, R2_e2, such, inhibition of RNR to control eukaryotic pathogens has offers additional unique regions for drug-targeted inhibition also been suggested (Dormeyer et al., 1997; Ekanem, 2001), (Munro et al., submitted). It is these differences between in particular those belonging to Apicomplexa (Chakrabarti et apicomplexan and mammalian host sequences that may al., 1993; Barker et al., 1996; Akiyoshi et al., 2002). In fact, best be exploited when designing chemotherapeutic drugs RNR was included in a set of 57 “gold standard” essential to specifcally target the Apicomplexa, making RNR an enzymes with experimentally documented antimalarial appealing option as a drug target. effects (Huthmacher et al., 2010). These, and other studies, have resulted in a considerable array of approaches to RNR has a long history as chemotherapeutical target inhibit RNR, which we briefy describe next. Much research has focused on the relationship between the class Ia R1 and R2 subunits in the context of human cancer. Methods of RNR inhibition It has been hypothesized that normal or over-expression of RNR may be targeted at the translational or protein levels. R1 results in suppression of malignant cells (Yen, 2003) RNR inhibitors are loosely categorized as those that prohibit and Fan et al. (Fan et al., 1997) demonstrated that the R1 the formation of an active holoenzyme or those that inhibit the

translation inhibitor prevent translation of mRNA  




  no subunits formed "
RNAi "
antisense oligonucleotides

subunits formed

dimerization inhibitor prevent holoenzyme formation 


  no holoenzyme formed "


 sequences

holoenzyme formed catalytic & allosteric inhibitors interfere with holoenzyme "
nucleoside analogues (R1) holoenzyme inactivated "
allosteric inhibitors (R1) holoenzyme "
  
 
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Figure 2. Means of RNR inhibition. A fowchart showing the progression from mRNA to the formation of the holoenzyme (ovals) and how translation, dimerization and catalytic and allosteric inhibitors act along this process. Ribonucleotide Reductase as a Target to Control Apicomplexan Diseases 13 function of an already fully-formed holoenzyme (Cerqueira the silencing of target genes with chemically synthesized et al., 2007). At the translation level, synthesis of the enzyme small interfering RNA (siRNA) (Lee and Sinko, 2006; Vaish subunits is blocked, while at the protein level, inhibitors may et al., 2010), which typically utilizes much shorter lengths be employed to prevent the formation of the holoenzyme of double stranded RNA (20 to 25 bp). siRNA has proven or inhibitors may be used to inactivate either the R1 or R2 useful in the suppression of p53R2 expression, leading to subunit, or both (Cerqueira et al., 2005) (Figure 2). The use the inhibition of tumor growth and an increase in sensitivity of ribozymes, single strand antisense oligonucleotides, and to anticancer drugs (Yanamoto et al., 2005). small interfering RNA (siRNA) can all be defned as anti- Antisense RNA has been documented in Plasmodium mRNA strategies, or subunit synthesis inhibitors (see (Aboul- (Militello et al., 2005) and there are numerous examples Fadl, 2005) for a review of the implementation, optimization, where RNAi has reportedly been successfully used to silence and practical application of these methodologies), while genes in Plasmodium (Kumar et al., 2002; Malhotra et al., dimerization, catalytic, and allosteric inhibitors focus on the 2002; McRobert and McConkey, 2002; Mohmmed et al., inhibition of formed proteins. 2003; Dasaradhi et al., 2005; Gissot et al., 2005; Crooke et al., 2006; Sunil et al., 2008; Tuteja et al., 2008; Sriwilaijaroen Subunit synthesis inhibitors et al., 2009). However, in stark contrast to these fndings, Ribozymes are those where support for RNAi in Plasmodium is lacking. Ribozymes are catalytic RNA molecules with distinct In fact, using both an experimental and bioinformatics three-dimensional confgurations, which principally exhibit approach, Baum et al. (Baum et al., 2009) suggested that trans-cleavage properties. Ribozymes can be specifcally Plasmodium lacks RNAi functionality and the conserved designed to cleave a targeted RNA sequence, thereby enzymes necessary for RNAi activity such as Dicer and inactivating gene transcripts (Haseloff and Gerlach, 1988; Argonaute-like proteins, or their analogs. It has further been Norris et al., 2000; Citti and Rainaldi, 2005). Ribozymes and suggested that documented RNAi activity in Plasmodium their applications have been extensively reviewed (Puerta- may be the result of general toxicity to the introduced RNA, Fernández et al., 2003; Nayak and Kohli, 2005; Khan, 2006; or an alternative (non-RNAi) antisense mechanism, and Tedeschi et al., 2009). Ribozymes offer a productive avenue not the result of specifc gene targeting by RNAi (Ullu et for gene therapy and have been designed for use against al., 2004). Additional authors have also questioned RNAi inborn metabolic disorders, viral infections, and cancer activity or the presence of a classical RNAi pathway in (Lewin and Hauswirth, 2001). Apicomplexa, particularly in Plasmodium (Aravind et al., Both in vitro and in vivo studies demonstrated promising 2003; Blackman, 2003; Cerutti and Casas-Mollano, 2006; use of ribozymes to target the survivin gene, which when Xue et al., 2008). Further calling into question the utility of expressed, leads to cell proliferation, typical in most human RNAi is the fact that these studies show down-regulation, carcinomas (Choi et al., 2003). Ribozymes designed to target but not the elimination, of gene function, and the degree mouse telomerase RNA were successfully administered to which protein expression is suppressed depends of a and systemically expressed in vivo, and served to reduce variety of factors (Brown and Catteruccia, 2006). the metastatic progression of B16_F10 murine melanoma metastases (Nosrati et al., 2004). In vitro targeting of RhoC RNA antisense oligonucleotide inhibitors by ribozymes showed reduction of invasiveness in human Antisense oligonucleotides (AOs) are short (10 to 30 breast cancer cells and thus demonstrated the utility of nucleotides), single strands of RNA or DNA that serve ribozymes in gene therapy (Lane et al., 2010). Similarly, to inhibit gene expression. The sequence of an AO is ribozymes designed for targeting specifc sites for cleavage complementary to a chosen target mRNA sequence, to in human telomerase RNA were demonstrated to be effective which it will bind via canonical Watson-Crick base pairing. in arresting cell growth and induction of spontaneous cell A variety of modifcations to antisense oligonucleotides apoptosis in colon cancer cells (Lu et al., 2011). may be employed to prevent degradation, increase In the context of apicomplexan control, ribozymes affnity and potency, and to reduce non-target toxicity were successfully used to reduce malarial viability up to (Chan et al., 2006; Sahu et al., 2007; Li et al., 2010). 55% when targeting P. falciparum-specifc inserts in the Further improvements come in terms of selective delivery carbamoyl-phosphate II synthetase gene (Flores et al., systems for oligonucleotides (Ming et al., 2010). AOs may 1997). Accordingly, C-terminus insertions present in the knockdown a target mRNA molecule by means of three R1 subunit enzyme of Plasmodium and Theileria offer sites distinct processes: (1) steric inhibition, where protein uniquely different from those of their hosts, which may be translation is inhibited once AOs are bound to the target specifcally targeted by ribozymes (Ingram and Kinnaird, mRNA, (2) the non-specifc endonuclease, ribonuclease H 1999). (RNase H), may be activated and catalyze the cleavage of a DNA/mRNA duplex; alternatively, (3) pre-mRNA targeting, RNA interference which includes inhibition of splicing, inhibition of the 5' cap RNA interference (RNAi) utilizes segments of double- formation, or de-stabilization of the pre-mRNA, would inhibit stranded RNA to interfere with gene expression and it mRNA maturation (Ho et al., 1996; Baker and Monia, 1999; usually relies on the enzyme Dicer and the RNA-induced Achenbach et al., 2003; Sahu et al., 2007). silencing complex (Scherr et al., 2004). Theoretically, the Genes encoding both the large R1 RNR subunit and chemotherapeutic applicability of RNAi, and that of variants small R2 subunit have been targeted with antisense inhibition. on the RNAi theme, is extensive; however, caution is Inhibition of expression of the Herpes simplex virus was advised as there are safety and specifcity concerns (Grimm achieved using AOs to target the R1 translation initiation and Kay, 2007). There have been recent advances in the site (Aurelian and Smith, 2000). The R2-directed AO, GTI- reduction of non-target effects and improved specifcity in 2040, has shown promising selectivity and specifcity in its 14 Munro and Silva antitumor activity against a variety of human cancers (Lee et et al., 1998; Guittet et al., 1999). Hydroxyurea was shown to al., 2003; Desai et al., 2005). AOs were designed to target stop DNA synthesis in P. falciparum (Inselburg and Banyal, both the R1 and R2 subunits in oropharyngeal KB cancer 1984). Improved control of malaria utilizing a combination cells; however, only the targeted inhibition of R2 expression therapy of erythropoietin and iron sulfate in conjunction with reduced enzyme activity and inhibited cell growth (Chen et hydroxeurea has been hypothesized (Saei and Ahmadian, al., 2000). 2009). In contrast to the scavenging of radicals, iron AOs have also proven to be effective against a variety of chelators, which may destroy or prevent the formation of gene products in Plasmodium (Rapaport et al., 1992; Barker the radical (Nyholm et al., 1993; Richardson, 2002; Hodges et al., 1998; Gardiner et al., 2000; Patankar et al., 2001; et al., 2004; Whitnall et al., 2006), do not necessarily cause Kyes et al., 2002; Noonpakdee et al., 2003; Gunasekera permanent inhibition. Iron chelators target cellular iron, et al., 2004). RNR activity was inhibited by targeting the leading to iron deprivation, which has been suggested to region surrounding the translation initiation codon with AO result in RNR inhibition (Pradines et al., 1996). Iron chelators phosphorothioates for the P. falciparum R2_e1 subunit, have been demonstrated to be effective against the P. (Chakrabarti et al., 1993). falciparum trophozoite and ring stages, which, unlike host cells, demonstrated a limited to irreversible loss of capacity Protein inhibitors for recovery after the chelator was removed (Lytton et al., Dimerization (polymerization) inhibitors 1994). Dimerization inhibitors bind to one or more partners in a Substrate analogs are also referred to as suicide protein-protein interaction and hence prevent formation inhibitors and were recently reviewed (Perez et al., 2010). of the holoenzyme. In the case of RNR, they are small They are recognized as “normal” ribonucleotide substrates; peptidomimetic sequences that mimic the R2 subunit however, their interaction with the holoenzyme's active C-terminal residues. As such, they competitively bind to the site leads to inactivation of the enzyme. A case in point hydrophobic cleft in the R1 subunit, to the exclusion of R2, is inactivation of the R1 active site by use of nucleoside- and prevent formation of the holoenzyme (Paradis et al., diphosphate analogs, which bind and result in alkylation 1988; Gaudreau et al., 1990; Yang et al., 1990; Cosentino of the protein (Pereira et al., 2004; Pereira et al., 2006). et al., 1991). It is the differences between host and parasite Note that some substrate analogs such as gemcitabine and R2 C-terminal sequences that have so far lent themselves tezacitabine have additional inhibitory effects on the R2 to specifc targeting of a parasite's RNR. subunit (Salowe et al., 1993; Shao et al., 2006). RNR enzyme activity was inhibited in the Herpes simplex As noted earlier, nucleoside-triphosphates bind to the virus with the introduction of a peptide that corresponded to allosteric effector sites and serve to either activate or inhibit the C-terminus amino acid residues of the viral R2 subunit the reduction of nucleoside diphosphates (Thelander and (Gaudreau et al., 1992; Liuzzi et al., 1994). In addition to Reichard, 1979). Allosteric effector analogs, which are the Herpes simplex virus, RNR dimerization inhibitors have typically nucleoside-triphosphate analogs, thus interact with been extensively studied in E. coli, hamster, mouse, yeast, the two R1 allosteric effector-binding sites, i.e. the A- and and human cells (Cohen et al., 1986; Dutia et al., 1986; S-sites. dATP normally acts as an inhibitor; however, some Climent et al., 1991; Cosentino et al., 1991; Fisher et al., deoxyadenosine analogs have an even more powerful 1993; Davis et al., 1994). affect due to their higher affnity for the R1 effector site Likewise, in the case of apicomplexans, it is the (Cory and Mansell, 1975; Harrington and Spector, 1991; difference in the C-terminal sequences of the R2 subunits Jeha et al., 2004; Shao et al., 2006). Interference of the between parasites and their hosts that may be best exploited allosteric binding sites will have an infuence on the activity by chemotherapeutic means (Chakrabarti et al., 1993; and substrate binding affnity of the R1 subunit. While the Fisher et al., 1993; Cerqueira et al., 2005). Peptidomimetic structure of the allosteric sites in the R1 subunit may be inhibitors based on the C-terminus of the small subunit have similar between the RNR of an eukaryotic parasite and that been proposed as a means of disrupting the formation of of its host, there are usually unique substitutions between the RNR heterodimer complex in P. falciparum (Bracchi- the two, which in turn may lead to differences in allosteric Ricard et al., 2005). In fact, targeting malarial RNR activity regulation. That is the case of the RNR of humans and of P. falciparum-infected erythrocytes was accomplished by of Trypanosoma brucei, the causative agent of sleeping use of synthetic peptidomimetic peptides, which prevented sickness (Hofer et al., 1997). Chakrabarti et al. (Chakrabarti binding of the R1 and R2 subunits (Rubin et al., 1993). et al., 1993) suggested that differences in the N-terminus sequence of the R1 subunit of P. falciparum might indicate Catalytic and allosteric inhibitors that it too utilizes a different allosteric regulation mechanism Catalytic protein inhibitors target the active RNR holoenzyme relative to humans. The authors suggested that conservation and may act on either the R1 or R2 subunits, or both. across other protozoans, in terms of the residues whose Catalytic inhibitors may function by a variety of means, be function it is to bind dTTP, indicates that they too may it: (1) the destruction of the R2 subunit radical by radical employ an allosteric regulation mechanism that differs from scavengers or iron chelators, (2) inactivation of the R1 mammalian hosts. Such a difference has the potential to subunit active site, or (3) via substrate/nucleoside analogs, be exploited via suicide substrate inhibitors or nucleoside thus primarily acting on the R1 subunit. Allosteric inhibitors, analogues (Ingram and Kinnaird, 1999). which are also nucleoside analogs, target the R1 effector binding sites. Challenges to using RNR to control apicomplexan Radical scavengers such as hydroxyurea, irreversibly parasites destroy the tyrosol radical of the R2 protein (Krakoff et al., First and foremost, the function of the apicomplexan-specifc 1968; Lepoivre et al., 1991; Szekeres et al., 1997; Fontecave R2_e2 subunit remains unknown and, in particular, whether Ribonucleotide Reductase as a Target to Control Apicomplexan Diseases 15 this subunit is essential to the formation of a functional Human R2_e1 RNR holoenzyme has yet to be determined. Munro et al. A 1.0 (submitted) have identifed considerable variability among apicomplexans in the amino acid residue that purportedly 0.5 S harbors the free radical in the R2_e2 subunit, as well as in probability additional residues of functional importance. For example, 0.0 NVFTLDADF the lack of conservation of one of the two phenylalanine F7 F1 residues in the C-terminus (Figure 3A) raises the possibility Apicomplexan R2_e1 of a difference in the interaction between the R2_e2 and 1.0 R1 subunits relative to that observed with R2_e1. Following C T the residue notation of Fisher et al. (Fisher et al., 1993), 0.5 V AD 1 7 S N L

probability L the C-terminal residues F and F appear to be particularly D IK N S 0.0 Q TD E infuential in dictating the interaction/binding-specifcity to F F 7 1 subsites of the R1 subunit (Pellegrini et al., 2000; Pender et F F al., 2001). While the R2_e2 C-terminal residue equivalent Apicomplexan R2_e2 to F1 is maintained as phenylalanine and conserved 1.0 7 K across R2_e2, the residue equivalent to F is instead S substituted for isoleucine in all sequences sampled, save 0.5 Q TF E L

probability D V DS the cryptosporidians. This may, however, not be a concern; SFLTNEQ 7 0.0 I it has been established that F need not be stringently DF F7 F1 conserved because the R1 subsite interacting with this R2 subunit position can accommodate a variety of hydrophobic R2_e2 residues (Pender et al., 2001; Gao et al., 2002; Cooperman, B Bab_bovis_XP_001610573 SISFNEDF Bab_equi_6m007985 EIKFDEDF 2003). Also, Tyr370 in mouse R2 was determined to be The_annulata_XP_953574 DIKFDEDF essential in the radical transport pathway (Rova et al., The_parva_XP_766717 EIKFDEDF Pla_berghei_Pdb_121420 QITFDEDF 1999) and yet an equivalent to this residue is lacking in the Pla_yoelii_XP_727957 QITFDEDF apicomplexan-specifc R2_e2 subunit. Pla_chabaudi_PCAS_121490 QITFDEDF There are further challenges to RNR chemotherapy. Pla_falciparum_XP_001347439 QILFDEDF Pla_knowlesi_XP_002258799 QIVLDEDF Two widely used anti-cancer RNR inhibitors, hydroxyurea Pla_vivax_XP_001614470 QIVLDEDF and gemcitabine, are toxic to humans (Banach and Williams, Cry_hominis_XP_665685 QFSTSQDF 2000; Santini et al., 2000). Additionally, resistance to RNR Cry_muris_XP_002140092 QFSTSQDF R2_e2 only .: .:** inhibition by some radical scavengers and iron chelators has been observed (Nocentini et al., 1990; Sneeden and Loeb, R2_e1 2004; Fu and Xiao, 2006). Further concerns with regard to Hom_sapiens_isoform_2_NP_001025(p53R2) NSFTLDADF Hom_sapiens_isoform_1_NP_056528 NVFTLDADF the use of ribozymes include method of delivery, ribozyme Bab_bovis_XP_001610982 QKFATDADF stability, the secondary and tertiary structure of the target Bab_equi_6m007342 QKFSTDIEF Pla_berghei_Pdb_103660 QVFSLNTDF mRNA, and how these factors relate to accessibility of the Pla_chabaudi_XP_739266 QVFSLNTDF region being targeted for cleavage (Turner, 2000). Pla_yoelii_XP_723858 QVFSLNTDF Pla_falciparum_XP_001348226 QVFCLNTEF Pla_reichenowi_novel_model_330 QVFCLNTEF Bolstering support for use of RNR inhibition to control Pla_gallinaceum_rna_PF_0053_1_1cds QVFCLNTDF apicomplexan parasites Pla_knowlesi_XP_002260936 QVFCLNSDF Since its discovery in 1961 (Reichard et al., 1961), Pla_vivax_XP_001616894 QVFCLNSDF Cry_hominis_XP_665115 QKFDLNADF ribonucleotide reductase has been featured in almost 5,500 Cry_parvum_XP_627447 QKFDLNADF publications in a variety of felds, such as biochemistry, Cry_muris_XP_002140093 QIFNLDAEF The_annulata_XP_954052 QKFSTDLEF molecular biology, oncology, cell biology, and chemistry. As The_parva_XP_766246 QKFSTDLEF such, there is a wealth of information regarding this protein. R2_e1 only : * : :* The literature is replete with studies of RNR inhibitor use in R2_e2 and R2_e1 : . :* the control of cancer and of human pathogens. As detailed earlier, RNR has also received attention for its potential Figure 3. RNR subunit R2 C-terminus. A) Sequence logo use as a target to control the apicomplexan parasite P. representation of human and apicomplexan R2_e1 and falciparum. RNR inhibitors have already been shown to apicomplexan R2_e2 terminal residues. F1 and F7 are crucial have some antimalarial activity. Examples include RNA phenylalanine residues that interact with the R1 subunit. antisense oligonucleotide inhibitors (Chakrabarti et al., The numbering of these residues follows (Fisher et al., 1993) and radical scavengers such as the substituted/ 1993), while subsequent authors appear to have reversed modifed benzohydroxamic acids, specifcally the vicinal the order (Pellegrini et al., 2000; Pender et al., 2001; Gao dihydroxybenzohydroxamates (Holland et al., 1998). et al., 2002; Cooperman, 2003). Created with WebLogo 3 Knowledge of protein structure and localization can (Schneider and Stephens, 1990; Crooks et al., 2004). B) greatly aid in the design of chemotherapeutic drugs. For Terminal residues for human and apicomplexan R2_e1 and example, it is essential to determine if the region being apicomplexan R2_e2 from which the logos were derived. targeted is exposed on the protein's surface, whether Data derived from Munro et al. (submitted). Plasmodium and it is functional, and whether the residues surrounding human sequences are underlined for comparative purposes the target region in its native conformation are similarly (see text). Amino acid substitutions: . = semiconserved, : = conserved (Durand et al., 2008). While the structure of the conserved, * = identical. 16 Munro and Silva apicomplexan-specifc R2 subunit is not known, structure of suited for chemotherapeutical intervention, since it is the the orthodox R2_e1 subunit from Plasmodium vivax (2O1Z) intraerythrocytic stages of Plasmodium that cause clinical and P. yoelii (2P1I) are deposited on the RCSB Protein Data symptoms (Yeh and Altman, 2006). Additionally, human Bank (www.pdb.org; Berman et al., 2000). RBCs are not nucleated, thus precluding an alternative Advances have been made in the production of means for the parasite to exploit host RNR (Rubin et al., apicomplexan recombinant proteins, a process that has 1993). been historically hampered by the A+T-biased nature of the The utility of RNR as an antimalarial/antipathogen target plasmodial genome and uncommon codon usage (Weber, depends upon the ability to specifcally target the pathogen's 1987; Anonymous, 2006; Brombacher 2006). However, RNR and not that of the host, as RNR is essential to both see Vedadi et al. (Vedadi et al., 2007) who found E. coli species. R1 and R2 subunits are highly conserved between to effectively produce apicomplexan proteins on a large- prokaryotes and eukaryotes in the regions containing the scale basis. Codon optimization (Hedfalk et al., 2008) functionally important residues (Chakrabarti et al., 1993; and codon harmonization (Hillier et al., 2005; Angov et al., Roshick et al., 2000; Voegtli et al., 2001; Högbom et al., 2008; Chowdhury et al., 2009) have been used to improve 2004; Högbom, 2010); however, they differ in the N- and expression of Plasmodium proteins in E. coli. Further C-terminal sequences (Ingram and Kinnaird, 1999). Novel advances have been made in the area of the use of a RNRs are additional potential targets for new drugs, phylogenetically similar, or “pseudoparasite”, expression especially if they provide distinct differences between host system (Fernádez-Robledo and Vasta, 2010). Furthermore, and parasite sequences. The necessity for target specifcity in vitro protocols for P. falciparum are established, although to avoid side, or non-target, effects in humans cannot in vivo animal models are based on the murine Plasmodium be overstated. The use of antisense oligonucleotides in species P. berghei, P. chabaudi, P. vinkei, and P. yoelii, and the control of some cancers has shown that the drugs in not those that parasitize humans (Fidock et al., 2004). question have favorable toxicity profles, in part because To a large degree, the utility of RNR as an antimalarial of their ability to specifcally target segments of RNA chemotherapeutic target is dependent upon the timing of (Davies et al., 2003). The unorthodox R2_e2 apicomplexan the protein's expression. In mammals and yeast, the large subunit provides a distinct and additional opportunity for subunit R1 has a half-life of 24 hours and is maintained specifc drug targeting. One example is the C-terminus of at a constant level throughout a cell's life cycle, while the the R2 subunit, which differs considerably between the small subunit R2 has a half-life of around 3 hours and its human R2_e1 subunits and both the R2_e1 and R2_e2 expression is restricted from the S-phase through to late apicomplexan subunits (Figures 3A and 3B). This difference mitosis, at which time it is rapidly degraded (Eriksson et al., in the C-terminal sequences of the R2 subunits between 1984; Engström et al., 1985; Björklund et al., 1990; Elledge apicomplexans and their hosts can be ideally targeted by et al., 1992). The non-canonical human p53R2 protein is chemotherapeutic means (Rubin et al., 1993; Fisher et al., expressed during periods of DNA repair (Håkansson et al., 1995; Ingram and Kinnaird, 1999). 2006). In mammalian cells, it has been demonstrated that it Finally, it is worth noting that Plasmodium-infected is the presence or absence of the R2 protein that regulates erythrocytes demonstrate an increase in cell membrane RNR activity (Chabes and Thelander, 2000). In contrast to permeability (Baumeister et al., 2011). In vitro uptake of this, in S. cerevisiae it is the R1 subunit whose transcription small pieces of RNA becomes less of an issue as RBCs that is increased during S-phase, thus controlling RNR activity are infected with malaria exhibit an enhanced and selective (Ortigosa et al., 2006). The utility of RNR inhibition in the uptake of such molecules in comparison to non-infected control of certain cancers has relied in part on the fact that RBCs (Rapaport et al., 1992), a difference attributed to RNR is most needed when cells are rapidly proliferating, the presence of a parasitophorous ducts in infected RBCs rendering cancerous cells particularly vulnerable to (Pouvelle et al., 1991). RNR inhibition (Smith and Karp, 2003). In P. falciparum, ribonucleotide biosynthesis begins as early as the ring Summary and early trophozoite stage; however, deoxyribonucleotide Extensive research has already established that RNR metabolism occurs later, with both R1 and R2_e1 subunit has potential as an antimalarial drug. What is particularly transcripts detected in the red blood cells (RBCs) at 10 appealing about RNR inhibition as a means of controlling hours post RBC infection and peaking 31 to 33 hours Apicomplexa is the potential control of not one, but two, post-infection, which coincides with the late trophozoite/ copies of the R2 subunit, both of which are distinct from that early schizont stage (Chakrabarti et al., 1993; Bozdech et of the host. Additionally, in the case of the malarial parasite al., 2003; Bozdech and Ginsburg, 2004). Comprehensive Plasmodium, RNR expression occurs from the sporozoite expression studies in P. falciparum show that the expression through the gametocyte life cycle stage, offering multiple profle of R2_e2, albeit less intense, matches that of the opportunities for chemotherapeutic targeting. This may well other to subunits (Bozdech et al., 2003; Llinás et al., 2006). be the case for other Apicomplexa parasites that undergo The timing of RNR expression is not surprising, since it is rapid clonal expansion in the host. during the intraerythrocytic stages that the malarial parasite Of the eight established methods of RNR inhibition undergoes logarithmic growth and requires RNR for DNA discussed, RNAi seems the least promising in terms of synthesis (Yeh and Altman, 2006). Additionally, expression controlling apicomplexan parasites as the necessary of PfR4 (R2_e2) has been detected in the sporozoite and enzymes appear to be lacking. Ribozyme approaches have gametocyte stages of the parasite life cycle (Bracchi-Ricard been successfully implemented in Plasmodium; however et al., 2005), and in the case of P. yoelii, both R1 and R2_ their use against RNR has not yet been demonstrated. In e1 transcripts were detected in the liver stage of infection contrast, substrate analogs and allosteric effector analogs (Nivez et al., 2000). These expression profles are well- have been effectively used to inhibit RNR, but their use Ribonucleotide Reductase as a Target to Control Apicomplexan Diseases 17 in Plasmodium has yet to be demonstrated. Antisense Aurelian, L., and Smith, C.C. (2000). Herpes simplex virus oligonucleotide inhibitors, dimerization inhibitors, radical type 2 growth and latency reactivation by cocultivation are scavengers, and iron chelators have all been successfully inhibited with antisense oligonucleotides complementary used to target RNR in Plasmodium. Ribozymes, antisense to the translation initiation site of the large subunit of oligonucleotide inhibitors, and dimerization inhibitors show ribonucleotide reductase (RR1). Antisense Nucleic Acid the most promise in terms of future anti-apicomplexan drug Drug Dev 10, 77-85. development. On the other hand, resistance to some radical Baker, B.F., and Monia, B.P. (1999). Novel mechanisms scavengers and iron chelators has been established, they for antisense-mediated regulation of gene expression. can not be use to selectively target a peptide, and the fact Biochim Biophys Acta 1489, 3-18. that the effects of iron chelators are generally reversible, Banach, M.J., and Williams, G.A. (2000). Purtscher makes them less appealing prospects. retinopathy and necrotizing vasculitis with gemcitabine The difference in sequence similarity between the therapy. Arch Ophthalmol 118, 726-727. parasite and human R2_e1 subunits is considerable, Barker, R.H., Metelev, V., Coakley, A., and Zamecnik, and the difference is even more accentuated when the P. (1998). Plasmodium falciparum: Effect of chemical parasite's R2_e2 subunit is considered (Munro et al., structure on effcacy and specifcity of antisense submitted). 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